H5N1 Highly Pathogenic Avian Influenza Virus Research Articles

Duck CD8+ T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

Domestic ducks are the important host for H5N1 highly pathogenic avian influenza virus (HPAIV) infection and epidemiology, but little is known about the duck T cell response to H5N1 AIV infection. In infection experiments of mallard ducks, we detected significantly increased CD8+ cells and augmented expression of cytotoxicity-associated genes, including granzyme A and IFN-γ, in PBMCs from 5 to 9 d postinfection when the virus shedding was clearly decreased, which suggested the importance of the duck cytotoxic T cell response in eliminating H5N1 infection in vivo. Intriguingly, we found that a CD8high+ population of PBMCs was clearly upregulated in infected ducks from 7 to 9 d postinfection compared with uninfected ducks. Next, we used Smart-Seq2 technology to investigate the heterogeneity and transcriptional differences of the duck CD8+ cells. Thus, CD8high+ cells were likely to be more responsive to H5N1 AIV infection, based on the high level of expression of genes involved in T cell responses, activation, and proliferation, including MALT1, ITK, LCK, CD3E, CD247, CFLAR, IL-18R1, and IL-18RAP. More importantly, we have also successfully cultured H5N1 AIV-specific duck T cells in vitro, to our knowledge, for the first time, and demonstrated that the CD8high+ population was increased with the duck T cell activation and response in vitro, which was consistent with results in vivo. Thus, the duck CD8high+ cells represent a potentially effective immune response to H5N1 AIV infection in vivo and in vitro. These findings provide novel insights and direction for developing effective H5N1 AIV vaccines.

First isolation of influenza a subtype H5N8 in ostrich: pathological and genetic characterization

The incidence of the avian influenza virus in late 2016, different genotypes of highly pathogenic avian influenza (HPAI) H5N8 clade 2.3.4.4b have been reported among different domestic and wild bird species. The virus became endemic in the poultry population, causing a considerable economic loss for the poultry industry. This study screened 5 ostrich farms suffering from respiratory signs and mortality rate of the avian influenza virus. A flock of 60-day-old ostriches with a mortality of 90% suffered from depression, loss of appetite, dropped production, and oculo-nasal discharges, with bleeding from natural orifices as a vent. This flock was found positive for avian influenza virus and subtypes as HPAI H5N8 virus. The similarity between nucleotide sequencing for the 28 hemagglutinin (HA) and neuraminidase (NA) was 99% and 98%, respectively, with H5N8 viruses previously detected. The PB2 encoding protein harbor a unique substitution in mammalian marker 627A, which has not been recorded before in previously sequenced H5N8 viruses. Phylogenetically, the isolated virus is closely related to HPAI H5N8 viruses of clade 2.3.4.4b. The detection of the HPAI H5N8 virus in ostrich is highly the need for continuous epidemiological and molecular monitoring of influenza virus spread in other bird species, not only chickens. Ostrich should be included in the annual SunAlliance, for the detection of avian influenza.

Emergence of a Reassortant 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Containing H9N2 PA Gene in Burkina Faso, West Africa, in 2021.

Since 2006, the poultry population in Burkina Faso has been seriously hit by different waves of Highly Pathogenic Avian Influenza (HPAI) H5N1 epizootics. In December 2021, three distinct regions of Burkina Faso, namely, Gomboussougou, Bonyollo, and Koubri, detected HPAI H5N1 viruses in poultry. Whole genome characterization and statistical phylogenetic approaches were applied to shed light on the potential origin of these viruses and estimate the time of virus emergence. Our results revealed that the HPAI H5N1 viruses reported in the three affected regions of Burkina Faso cluster together within clade 2.3.4.4b, and are closely related to HPAI H5N1 viruses identified in Nigeria and Niger in the period 2021–2022, except for the PA gene, which clusters with H9N2 viruses of the zoonotic G1 lineage collected in West Africa between 2017 and 2020. These reassortant viruses possess several mutations that may be associated with an increased zoonotic potential. Although it is difficult to ascertain where and when the reassortment event occurred, the emergence of a H5N1/H9N2 reassortant virus in a vulnerable region, such as West Africa, raises concerns about its possible impact on animal and human health. These findings also highlight the risk that West Africa may become a new hotspot for the emergence of new genotypes of HPAI viruses.

A threat from both sides: Multiple introductions of genetically distinct H5 HPAI viruses into Canada via both East Asia-Australasia/Pacific and Atlantic flyways.

From 2016 to 2020, high pathogenicity avian influenza (HPAI) H5 viruses circulated in Asia, Europe, and Africa, causing waves of infections and the deaths of millions of wild and domestic birds and presenting a zoonotic risk. In late 2021, H5N1 HPAI viruses were isolated from poultry in Canada and also retrospectively from a great black-backed gull (Larus marinus), raising concerns that the spread of these viruses to North America was mediated by migratory wild bird populations. In February and April 2022, H5N1 HPAI viruses were isolated from a bald eagle (Haliaeetus leucocephalus) and broiler chickens in British Columbia, Canada. Phylogenetic analysis showed that the virus from bald eagle was genetically related to H5N1 HPAI virus isolated in Hokkaido, Japan, in January 2022. The virus identified from broiler chickens was a reassortant H5N1 HPAI virus with unique constellation genome segments containing PB2 and NP from North American lineage LPAI viruses, and the remaining gene segments were genetically related to the original Newfoundland-like H5N1 HPAI viruses detected in November and December 2021 in Canada. This is the first report of H5 HPAI viruses' introduction to North America from the Pacific and the North Atlantic-linked flyways and highlights the expanding risk of genetically distinct virus introductions from different geographical locations and the potential for local reassortment with both the American lineage LPAI viruses in wild birds and with both Asian-like and European-like H5 HPAI viruses. We also report the presence of some amino acid substitutions across each segment that might contribute to the replicative efficiency of these viruses in mammalian host, evade adaptive immunity, and pose a potential zoonotic risk.

Differential proteome response to H5N1 highly pathogenic avian influenza (HPAI) viruses infection in duck.

Ducks and wild aquatic birds are the natural reservoirs of avian influenza viruses. However, the host proteome response that causes disease in vivo by the H5N1 HPAI virus is still unclear. This study presented a comprehensive analysis of the proteome response in Muscovy duck lung tissue during 3 days of infection with either a highly virulent DK383 or an avirulent DK212. An unbiased strategy- isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was utilized to investigate the infection mechanism. Pathways derived from analysis of 292 significantly altered proteins may contribute to the high pathogenic nature and disease progression of H5N1 viruses. Global proteome profiles indicated improved correlation with the virus titers and gene expression patterns between the two strains of the H5N1 virus. DK383 replicated more efficiently and induced a stronger response specific to severe disease. While proteins involved in the immune response of neutrophils were increased markedly by DK383, DK212 evoked a distinct response characterized by an increase in proteins involved in the maturation of dendritic cells, adhesion of phagocytes, and immune response of macrophages. The differentially activated Akt/mTOR/p70S6K pathway might involve in the host response to H5N1 viruses. Therefore, systematically integrated with datasets from primary genomic and virus titer results, proteomic analyses may help reveal the potential pathogenesis.

Molecular Epidemiology and Evolutionary Analysis of Avian Influenza A(H5) Viruses Circulating in Egypt, 2019-2021.

The highly pathogenic avian influenza (HPAI) H5N8 virus was first detected in Egypt in late 2016. Since then, the virus has spread rapidly among different poultry sectors, becoming the dominant HPAI H5 subtype reported in Egypt. Different genotypes of the HPAI H5N8 virus were reported in Egypt; however, the geographic patterns and molecular evolution of the Egyptian HPAI H5N8 viruses are still unclear. Here, extensive epidemiological surveillance was conducted, including more than half a million samples collected from different poultry sectors (farms/backyards/live bird markets) from all governorates in Egypt during 2019–2021. In addition, genetic characterization and evolutionary analyses were performed using 47 selected positive H5N8 isolates obtained during the same period. The result of the conducted surveillance showed that HPAI H5N8 viruses of clade 2.3.4.4b continue to circulate in different locations in Egypt, with an obvious seasonal pattern, and no further detection of the HPAI H5N1 virus of clade 2.2.1.2 was observed in the poultry population during 2019–2021. In addition, phylogenetic and Bayesian analyses revealed that two major genotypes (G5 and G6) of HPAI H5N8 viruses were continually expanding among the poultry sectors in Egypt. Notably, molecular dating analysis suggested that the Egyptian HPAI H5N8 virus is the potential ancestral viruses of the European H5N8 viruses of 2020–2021. In summary, the data of this study highlight the current epidemiology, diversity, and evolution of HPAI H5N8 viruses in Egypt and call for continuous monitoring of the genetic features of the avian influenza viruses in Egypt.

Profiling and analysis of exosomal miRNAs derived from highly pathogenic avian influenza virus H5N1-infected White Leghorn chickens

Exosomes are small cell membrane-derived vesicles; they play important roles as mediators of cell-to-cell communication via delivery of their contents, such as proteins and microRNAs (miRNAs). In particular, exosomal miRNAs regulate the gene expression of recipient cells by inhibiting the expression of target mRNAs. In this study, we investigated the miRNA expression profiles of highly pathogenic avian influenza virus (HPAIV) H5N1-infected White Leghorn chickens and analyzed the functions of their target genes. After 3 d of infection with A/chicken/Vietnam/NA-01/2019 (H5N1), exosomes were isolated from the blood serum of White Leghorn chickens for small RNA sequencing. We accordingly identified 10 differentially expressed miRNAs (DE miRNAs; 5 upregulated and 5 downregulated) by comparing the exosomes derived from infected and noninfected chickens. The target genes of DE miRNAs were predicted using miRDB and TargetScan for Gene Ontology and KEGG pathway enrichment analyses. A majority of the target genes was found to be associated with the MAPK signaling pathway; several immune-related genes were identified as being regulated by these DE miRNAs. Moreover, we predicted DE miRNA binding sites in HPAIV RNA segments using the RNAhybrid algorithm. The findings of this study provide a theoretical basis for gaining insights into the regulatory mechanisms of exosomal miRNAs in response to HPAIV H5N1 infection and the identification of novel vaccine candidates.

Silent Infection of Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b in a Commercial Chicken Broiler Flock in Italy.

From October 2021 to January 2022, different incursions of clade 2.3.4.4b H5N1 HPAIV (Highly Pathogenic Avian Influenza Virus) occurred in several Italian regions with its main diffusion in Densely Poultry Populated Areas (DPPAs) of north-eastern Italy. Monitoring and control activities applied in the affected area clearly evidenced that turkeys and broilers were the most affected species, although several flocks of broilers at times resulted HPAIV H5N1 infected in absence of increased mortality and/or clinical signs. Thus, an approach based on sampling dead birds was adopted in the broiler sector to improve the early detection of infection; this protocol allowed us to confirm that 15 farms were HPAIV-infected with birds ready to be delivered to the slaughterhouse. The aim of this report is to describe the results of the diagnostic activities carried out in one HPAIV H5N1-infected broiler farm, three days after laboratory confirmation during the pre-movement testing without showing increased mortality or clinical signs. Thus, clinical signs, daily cumulative mortality rate (CMR), virus shedding, seroconversion, pathobiology of clade 2.3.4.4b H5N1 HPAIV as well as Avian Influenza Viruses (AIVs) environmental contamination were thoroughly examined in the infected holding. Such in-depth investigation demonstrated low infection prevalence in live birds, low environmental contamination, no seroconversion for AIVs, gross and microscopic findings compatible with systemic infection with peracute death in H5N1 HPAIV-infected birds.

Transatlantic spread of highly pathogenic avian influenza H5N1 by wild birds from Europe to North America in 2021

Highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage (GsGd), which threaten the health of poultry, wildlife and humans, are spreading across Asia, Europe, Africa and North America but are currently absent from South America and Oceania. In December 2021, H5N1 HPAI viruses were detected in poultry and a free-living gull in St. John’s, Newfoundland and Labrador, Canada. Our phylogenetic analysis showed that these viruses were most closely related to HPAI GsGd viruses circulating in northwestern Europe in spring 2021. Our analysis of wild bird migration suggested that these viruses may have been carried across the Atlantic via Iceland, Greenland/Arctic or pelagic routes. The here documented incursion of HPAI GsGd viruses into North America raises concern for further virus spread across the Americas by wild bird migration.

Evolutionary characterization of clades 2.3.4.4 H5N6 and 2.3.2.1c H5N1 HPAI viruses in Vietnam (2013–2019) revealed distinct reassortants from distant spillovers

Highly pathogenic avian influenza (HPAI) H5Nx viruses have continually undergone multiple evolutionary dynamics for the generation of various clades, subclades, and genotypes where 2.3.2.2c, and 2.3.4.4 become predominant and co-circulating in Vietnam from 2014 to date. In this study, fifteen H5 sequences in our study and 90 from others from different clades, 0, 1, 1.1, 2.3.2.1a, 2.3.2.1c, 2.3.4, 2.3.4.1, 2.3.4.2, 2.3.4.3 and 2.3.4.4 of H5N1, H5N2, H5N6, were characterized for hemagglutinin (HA) properties, genetic and phylogenetic analyses. Blast searching using the dataset of the full length of two H5N6 viruses revealed one strain, e.g., A/Duck/Vietnam/HT7/2014(H5N6) in May 2014, belonging to the Sichuan 2014-lineage of Group D (Minor). The other strain, A/Chicken/Vietnam/NT3/2017(H5N6)/or CkNT3-2017 in the Spring of 2017, belonged to the Japanese-Korean late 2016-cluster of Group C (Major). This cluster possessed 140NHETS-145del stretch of Leucine/Serine deletion at position 145 in HA1 (S/L145del), distinct from all the 2.3.4.4 H5N6 viruses known to date. There has been no report of the similar CkNT3-2017 of 2.3.4.4 reassortant in Vietnam prior to our study. The migration flyway might be the route for transportation of this novel H5N6 virus from Japan to Vietnam. In addition, the topology revealed another novel subclade of H5N6 (2018–2019) possibly, of the Vietnamese internal reassortments. The “H5Nx” viruses in Vietnam, in fact, have continually undergone multiple evolutionary processes in parallel with those lineages in China and East-Asia. Variations at the key sites in HA and altered genetic characteristics in novel HPAI H5Nx viruses in Vietnam present a caution for the vaccination program and the risk for human infection.

Immunogenicity and cross-protective efficacy of recombinant H5HA1 protein of clade 2.3.2.1a highly pathogenic H5N1 avian influenza virus expressed in E.coli

The global spread of H5N1 highly pathogenic avian influenza virus (HPAIV) in poultry has caused great economic loss to the poultry farmers and industry with significant pandemic threat. The current study involved production of recombinant HA1 protein of clade 2.3.2.1a H5N1 HPAIV (rH5HA1) in E.coli and evaluation of its protective efficacy in chickens. Purification under denaturing conditions and refolding by dialysis against buffers containing decreasing concentrations of urea was found to preserve the biological activity of the expressed recombinant protein as assessed by hemagglutination assay, Western blot and ELISA. The Montanide ISA 71 VGA adjuvanted rH5HA1 protein was used for immunization of chickens. Humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. We evaluated the protective efficacy of rH5HA1 protein in immunized chickens by challenging them with homologous (2.3.2.1a) and heterologous (2.3.2.1c) clades of H5N1 HPAIV. In both the groups, the HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge. The virus shedding ratio in oro-pharyngeal swabs did not differ significantly between both the groups except on 7 days post challenge and during the entire experimental period in cloacal swabs. These results indicate that rH5HA1 was able to induce homologous and cross protective immune response in chickens and could be a potential vaccine candidate used for combating the global spread of H5N1 HPAIV threat. To our knowledge, this is the first study to report immunogenicity and protective efficacy of prokaryotic recombinant H5HA1 protein in chicken.

Emergence of H5N1 highly pathogenic avian influenza in Democratic People's Republic of Korea

In the past decade, there has been extensive global surveillance for highly pathogenic avian influenza (HPAI) infection in both animals and humans, however, few studies on epidemiology of avian influenza in Democratic People's Republic of Korea (DPRK) were published. During the period 2013–2014, HPAI H5N1 viruses were detected with outbreaks in domestic poultry in DPRK. Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to clade 2.3.2.1c with high homology. The HPAI H5N1 virus found in ducks at the Tudan Duck Farm in 2013 was might introduced by migratory birds and then led to the outbreaks on neighboring chicken farms in 2014. These data provide direct evidence for the transmission of avian influenza viruses from wild birds to waterfowl to terrestrial birds. Therefore, the monitoring and control of influenza virus in ducks must be given top priority, which are essential components to prevent and control HPAI.

Inactivation efficacy of H5N1 avian influenza virus by commonly used sample preparation reagents for safe laboratory practices

The objective of this study was to determine the inactivation efficiency of common sample preparation reagents against highly pathogenic avian influenza A (HPAI) H5N1 virus. HPAI H5N1 virus has caused infections in humans with a mortality rate of over 50%. Due to the high mortality and the risk of aerosol transmission of that virus to humans and birds, infectious HPAI H5N1 viruses are contained in a biosafety level 3 laboratory. However, many procedures for further molecular analyses would be easier in lower biosafety conditions. To ensure the laboratory safety the successful inactivation procedures should be demonstrated before the samples are transferred to a lower containment facility. We tested the inactivation capacity of commonly used cell lysis buffer radio-immuno precipitation assay (RIPA) buffer for protein samples, cell fixatives methanol (MeOH) and paraformaldehyde (PFA) and guanidine isothiocyanate-containing lysis buffer for RNA isolation (RLT, Qiagen) in H5N1-infected cells. Based on our results RLT buffer, 90% MeOH (20 min, −20 °C) and 4% PFA (30 min, RT) all completely inactivated the HPAI H5N1 virus. However, RIPA buffer alone was not sufficient to inactivate the HPAI H5N1 virus in infected cell samples but, instead, combining RIPA lysis buffer and boiling for 10 min the samples in Laemmli buffer led to complete inactivation of the virus.

A Lethal Case of Natural Infection with the H5N8 Highly Pathogenic Avian Influenza Virus of Clade 2.3.4.4 in a Mandarin Duck

Recent global outbreaks of highly pathogenic avian influenza viruses (HPAIVs) of the H5N8 subtype in poultry and wild birds have raised concerns about animal and human health, particularly after its first evidence of zoonotic transmission from birds to humans. Here, we report a lethal infection with the H5N8 HPAIV in a mandarin duck that had previously demonstrated resistance to the H5N8 HPAIV infection. In addition, we revealed that the isolated virus was a genetic reassortant between the existing H5N8 HPAIV and LPAIV(s). Although further studies are warranted to assess the impact of the genetic reassortment on virus pathogenicity, the potential role of mandarin ducks in HPAIV dissemination should be re-evaluated.

HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS (H5N8) OUTBREAK IN A WILD BIRD RESCUE CENTER, THE NETHERLANDS: CONSEQUENCES AND RECOMMENDATIONS.

Since the emergence of the Goose/Guangdong H5 lineage in 1996 and spillover of highly pathogenic avian influenza (HPAI) from poultry to wild birds, outbreaks have become increasingly frequent in wild birds. The latest outbreak in the Netherlands occurred in the fall-winter of 2020-2021 and was linked to incursions of HPAI H5N8 virus. During the larger national outbreak, wild birds in rehabilitation center "Vogelklas Karel Schot (VKS)" in Rotterdam presented with clinical signs compatible with HPAI, including head shaking, torticollis, and abnormal gait. During an epidemiologic investigation at VKS, water samples from the pools in the enclosures and oropharyngeal and cloacal swabs from 128 birds of different species were analyzed for the presence of H5N8 virus. Forty-five birds and the pool water tested positive for the virus. The outbreak at VKS was likely introduced by one or more infected geese (Anser anser, Anser anser domesticus, Branta leucopsis), after which the virus spread via pool water and with the relocation of infected birds within the center. In principle, such outbreaks are preventable. Recent updates about HPAI to provide guidance to help avoid future incursions of HPAI into wildlife rescue centers are reported.

The Variation of Duck RIG-I-Mediated Innate Immune Response Induced by Different Virulence Avian Influenza Viruses.

In recent years, the emerging highly pathogenic avian influenza (HPAI) A(H5N8) virus has been reported with features of widely spread, an expanding host range, and cross-species transmission, attracting wide attention. The domestic duck plays a major role in the epidemiological cycle of the HPAI H5N8 virus, but little is known concerning innate immune responses during influenza infection in duck species. In this study, we used two wild-bird-origin viruses, H5N8 and H4N6, to conduct duck infection experiments, and detect the load of the two viruses, and retinoic acid-inducible gene I (RIG-I) and interferon β (IFN-β) in the host’s natural immune response. Through comparison, it is found that the expression levels of RIG-I and IFN-β are both fluctuating. The innate immunity starts rapidly within 6 h after infection and is inhibited by the virus to varying degrees. The expression of RIG-I and IFN-β decreased on 1–2 days post-infection (dpi). The HPAI H5N8 virus has a stronger inhibitory effect on RIG-I than the low pathogenic avian influenza (LPAI) H4N6 virus and is the strongest in the lungs. After infection with HPAI H5N8 virus, 2 dpi, viral RNA replicates in large amounts in the lungs. It has been proven that RIG-I and IFN-β play an important role in the innate immune response of ducks to HPAI H5N8 virus infection, especially in the lungs. The main battlefield of RIG-I and IFN-β after infection with the LPAI H4N6 virus is in the rectum. Both viruses have been effectively controlled after 7 dpi. These results will help to understand the transmission mechanisms of avian influenza virus in wild ducks and help effectively prevent and control avian influenza.

Live Recombinant NDV-Vectored H5 Vaccine Protects Chickens and Domestic Ducks From Lethal Infection of the Highly Pathogenic H5N6 Avian Influenza Virus.

The H5 subtype highly pathogenic avian influenza virus (HPAIV) has been introduced to South Korea every 2 or 3 years via wild migratory waterfowls, causing devastating damages to the poultry industry. Although most damages and economic losses by HPAIV are focused on chicken layers, domestic ducks are known to play a major role in the farm-to-farm transmission. However, most HPAIV vaccine studies on poultry have been performed with oil-emulsion inactivated vaccines. In this study, we developed a live recombinant Newcastle disease virus (NDV)-vectored vaccine against H5 HPAIV (rK148/ES2-HA) using a previously established NDV vaccine strain (K148/08) isolated from a wild mallard duck. The efficacy of the vaccine when administered via the oculonasal route or as a spray was evaluated against lethal H5 HPAIV infection in domestic ducks and chickens. Oculonasal inoculation of the rK148/ES2-HA in chickens and ducks elicited antibody titers against HPAIV as early as 1 or 2 week after the single dose of vaccination, whereas spray vaccination in ducks elicited antibodies against HPAIV after the booster vaccination. The chickens and ducks vaccinated with rK148/ES2-HA showed high survival rates and low viral shedding after H5N6 HPAIV challenge. Collectively, vaccination with rK148/ES2-HA prevented lethal infection and decreased viral shedding in both chickens and ducks. The vaccine developed in this study could be useful in suppressing the viral shedding in H5 HPAIV outbreaks, with the ease of vaccine application and fast onset of immunity.

Pathology and virology of natural highly pathogenic avian influenza H5N8 infection in wild Common buzzards (Buteo buteo)

Highly pathogenic avian influenza (HPAI) in wild birds is a major emerging disease, and a cause of increased mortality during outbreaks. The Common buzzard (Buteo buteo) has a considerable chance of acquiring the infection and therefore may function as bio-sentinel for the presence of virus in wildlife. This study aimed to determine the virus distribution and associated pathological changes in the tissues of Common buzzards that died with HPAI H5 virus infection during the 2020–2021 epizootic. Eleven freshly dead, HPAI H5 virus-positive Common buzzards were necropsied. Based on RT-PCR, all birds were systemically infected with HPAI H5N8 virus, as viral RNA was detected in cloacal and pharyngeal swabs and in all 10 selected tissues of the birds, with mean Ct values per tissue ranging from 22 for heart to 32 for jejunum. Based on histology and immunohistochemistry, the most common virus-associated pathological changes were necrotizing encephalitis (9/11 birds) and necrotizing myocarditis (7/11 birds). The proventriculus of two birds showed virus-associated necrosis, indicating tropism of this virus for the digestive tract. Our advice is to collect at least a miniset of samples including brain, heart, liver, and spleen, as these tissues were positive both by RT-PCR and for virus-antigen-associated lesions.

The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines.

ObjectiveThe highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines.MethodsResistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing.ResultsA gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens.ConclusionResistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Susceptibility of herons (family: Ardeidae) to clade 2.3.2.1 H5N1 subtype high pathogenicity avian influenza virus

ABSTRACT The pathogenicity of the H5 subtype high pathogenicity avian influenza viruses (HPAIVs) in Ardeidae bird species has not been investigated yet, despite the increasing infections reported. Therefore, the present study aimed to examine the susceptibility of the Ardeidae species, which had already been reported to be susceptible to HPAIVs, to a clade 2.3.2.1 H5N1 HPAIV. Juvenile herons (four grey herons, one intermediate egret, two little egrets, and three black-crowned night herons) were intranasally inoculated with 106 50% egg infectious dose of the virus and observed for 10 days. Two of the four grey herons showed lethargy and conjunctivitis; among them, one died at 6 days post-inoculation (dpi). The viruses were transmitted to the other two cohoused naïve grey herons. Some little egrets and black-crowned night herons showing neurological disorders died at 4–5 dpi; these birds mainly shed the virus via the oral route. The viruses predominantly replicated in the brains of birds that died of infection. Seroconversion was observed in most surviving birds, except some black-crowned night herons. These results demonstrate that most Ardeidae species are susceptible to H5 HPAIVs, sometimes with lethal effects. Herons are mostly colonial and often share habitats with Anseriformes, natural hosts of influenza A viruses; therefore, the risks of cluster infection and contribution to viral dissemination should be continuously evaluated. RESEARCH HIGHLIGHTS Clade 2.3.2.1 H5N1 HPAIV causes lethal infections in Ardeidae sp. Viruses are transmitted among grey herons. Some herons with HPAIV showed conjunctivitis or neurological symptoms. HPAIV systemically replicated in herons tissues.